The bispecific antibody (BiAb) is an artificial antibody containing two specific antigen binding sites and can build a bridge between a target cell and a functional molecule (cell) to generate an oriented effector function. The BiAb has a broad application prospect in the biomedicine, especially in immunotherapy of tumors. To kill tumor cells through the BiAb-mediated cytotoxic effect is a hotspot of current application research of immunotherapy, and its principal characteristic lies in that the BiAb can simultaneously bind to a tumor-associated antigen and a target molecule on an immunologic effector cell and directly trigger the specific killing effect of the immunologic effector cell on the tumor cell. Immune cell antigens and tumor cell antigens under study and some background arts of related technology development will be introduced below.
1. CD3
The CD3 module consists of four subunits δ, ε, γ and ζ of which the molecular masses are 18.9 kDa, 23.1 kDa, 20.5 kDa and 18.7 kDa respectively and which have 171, 207, 182 and 164 amino acid residues in the length direction respectively. All the subunits constitute six peptide chains which tightly bind to a T cell receptor (TCR) usually to form a TCR-CD3 complex containing eight peptide chains (as shown in structural schematic diagram 1). This complex has the functions of transducing a T cell activation signal and stabilizing a TCR structure. The cytoplasmic domain of CD3 contains an immunoreceptor tyrosine-based activation motif (ITAM), and the TCR identifies and binds to an antigen peptide presented by an MHC (major histo-compatibility complex) molecule, resulting in that a tyrosine residue in a conserved sequence of the ITAM of CD3 is phosphorylated by tyrosine protein kinase p561ck in a T cell and then other tyrosine protein kinases (such as ZAP-70) containing an SH2 (Scr homology 2) structural domain can be collected. The phosphorylation of ITAM and the binding to ZAP-70 are one of important biochemical reactions in the early stage of the T cell activation signal transduction process. Therefore, the CD3 molecule has the function of transducing the activation signal generated when the TCR recognizes antigens.
2. HER2
Shih, et al. in 1981 (Shih C, Padhy L C, Murray M, et al. Transforming genes of carcinomas and neuroblastomas introduced into mouse fibroblasts [J]. Nature, 1981, 290(5803):261-264) cloned oncogene neu from mouse neuroblastoma genomes for the first time, and Slamon, et al. (1987, Science 2; 35; 177-182) separated the HER2 gene from a human cDNA library. It was found in the following sequence analysis and chromosome spectrum analysis that neu and HER2 are of the same gene which is customarily called as a HER2/neu gene or c-erbB-2 gene. HER2 is the 2rd member in the human epidermal growth factor receptor family belonging to the type I receptor tyrosine kinase family (also known as the ErbB receptor family) and takes an important regulating effect in growth, differentiation and metastasis processes of many normal and abnormal epidermic cells, and incidence, development and illness state of many tumors are closely related to the activity of HER2. There are a total of four receptors, namely HER1, HER2, HER3 and HER4 in the family. These receptors may interact to generate a heterogenetic or homologous dimer and activate multiple signal transduction pathways in cells, wherein the HER2 plays an important role in the signal transduction process of cells. The HER2 structurally comprises a binding domain of extracellular growth factors, a lipophilic transmembrane domain and an intracellular domain with regulatory carboxyl terminal fragments. The intracellular domain of the HER2 receptor has protein tyrosine kinase (PTK) activity and also possesses a number of tyrosine residue Tyr phosphorylation sites itself. The specific growth factor can induce the dimerization and stimulate the cross phosphorylation of the receptor after binding to the HER2 receptor, and the phosphorylated receptor can transduce an extracellular growth signal into the nucleus rapidly, and stimulate and control cell division-related genetic expression.
The HER2 which is positioned in human chromosome 17q21 and encodes a transmembrane protein having the molecular weight being 185 kD has the tyrosine kinase RTK activity, is at an inactive state under a normal condition, participates in regulation of normal differentiation of cells, is generally expressed only in the fetus stage and is only weakly expressed in a very few of normal tissues in an adult stage. The HER2 gene in the normal cell is a two-copy gene and can be activated through gene mutation, and its amplification will cause transcription up-regulation and protein synthesis increase, thus inhibiting tumor cell apoptosis, accelerating tumor cell proliferation, up-regulating vascular endothelial growth factor VEGF/vascular permeability factor VPF, accelerating tumor angiogenesis, increasing invasive ability of tumor cells and destroying anti-invasion bathers of body tissues, etc [Artufel M V, Valero A C, Llado R R, et al. Molecular Protocol for Her-2/neu analysis in breast carcinoma [J]. Clin Transl Oncol, 2005, 7. (11):504-511.]. The overexpression of the HER2 protein also plays an important role in inducing division, proliferation and transformation of cells and accelerating metastasis, invasion and adhesion of tumors [Hynes N E, Stem D F. The biology of erbB-2/Neu/HER-2 and its role in cancer [J]. Biochem Biophys AcTa, 1994, 1198 (2-3):165-184.].
Except for gene mutation or amplification, up-regulation of the HER2 expression may activate two main signal transduction pathways, namely an MAPK pathway and a PI3K/Akt pathway at the downstream of HER2, thus giving rise to a waterfall type chain reaction, regulating apoptosis-related genes, accelerating infinite proliferation and differentiation of cells, inhibiting apoptosis and further generating cancerization. The former pathway mainly participates in mitosis of cells and the latter pathway mainly affects survival and apoptosis of cells. The HER2 can activate the Ets transcription factor family member ER81 through the MAPK pathway to up-regulate human telomerase terminal transferase reverse transcriptase hTERT, which further causes abnormal activation of the telomerase terminal transferase of cells, so that the cells are transformed to enter a permanent proliferation state. [Goueli B S, Janknecht R. Upregulation of the catalytic telomerase subunit by the transcription factor ER81 and oncogenic HER2/Neu, Ras, or Raf. Mol Cell Biol, 2004, 24:25-35.]. After being activated, PI3K can catalyze phosphatidyl inositol PI to generate PIP2 and PIP3 which are important second messengers in a cell and can activate downstream protein kinase Akt/PKB to further cause phosphorylation of downstream BAD protein, thus preventing BAD and apoptosis proteins Bcl-2 and Bcl-XL from constituting a complex and simultaneously inducing the phosphorylation of forkhead transcription factors 1 to further inhibit the expression of apoptosis protogene.
Moreover, the HER2 oncogene is also a tumor metastasis driving factor, and the overexpression of the HER2 can increase the tumor cell metastasis capability through starting multiple metastasis-related mechanisms, such as cell migration rate, in vitro invasiveness and W-type collagenase activity and also can influence synthesis of certain adhesion molecules, such as epithelial cell E-cadherin, thereby accelerating metastasis. Carter, et al (Carter W, Hoying J, Boswell C, et al. HER-2/neu over-expression induces endothelial cell retraction [J]. Int Cancer, 2001, 91(3): 295-299), under study, considered that the over-expression of HER2 can induce endothelial cell retraction and dilated intercellular space, and it is easy for tumor cells to pass through among endothelial cells to generate shift or metastasis. Most studies considered that the HER2 gene amplification and/or protein over-expression were/was often a cue for us that the tumor malignancy is high and the metastasis ability is strong.
The over-expression of HER2 is often related to occurrence of tumors, for example:
(1) Gastric cancer: the gastric cancer is one of the most common malignant tumors in China, its prognosis is poor, the five-year survival rate of the gastric cancer in the development period is 5-20% only and the median survival time thereof does not exceed one year. The over-expression rate variation of the HER2 protein in the gastric cancer is detected, by different research groups, to be 7-43%. Positive expression of the HER2 protein in the gastric cancer is related to the tumor differentiation degree, Lauren typing and WHO typing and is not related to age, gender, tumorigenesis portion and clinical stages.
(2) Breast cancer: researches indicated that the HER2 generated gene amplification and protein over-expression in 20-30% of primary breast infiltrating ductal carcinoma. High expression of the HER2 always causes malignant metastasis of cells, and therefore, the HER2-positive breast cancer has strong infiltrability, short disease free survival time and poor prognosis. In vitro experiments displayed that apoptosis of tumor cells can be caused by inhibiting the expression of HER2.
(3) Ovarian cancer: the ovarian cancer is a main reason for gynecological tumor death. The over-expression of HER2 in the ovarian cancer is similar to that in the breast cancer, which accounts for 15-30%. Studies of Verri, et al (Verri, E, Guglielmini P, Puntoni M, et al. HER2/neu oncoprotein overexpression in epithelial ovarian cancer: evaluation of its prevalence and prognostic significance [J]. Oncology, 2005, 68:154-161) indicated that the total survival time of an HER2-positive (2+/3+) patient was remarkably shortened compared with that of an HER2-negative patient (0/1+) (29 months vs 48 months, P<0.05). The over-expression of the HER2 was found respectively through observing 20 cell lines from ovary in III and IV ovarian cancers.
(4) Prostate cancer: the genesis of the prostate cancer is androgen-dependent, and tumors will be retracted after medicinal or surgical castration, but will be finally changed to be androgen-independent to continuously grow, which is the most primary problem in the current treatment of the prostate cancer. Studies indicated that the HER2 is a main mediator of the prostate cancer in the process of transforming from androgen dependence to androgen independence. Signoretti, et al (Signoretti S, Montironi R, Manola J, et al. Her-2-neu expression and progression toward androgen independence in human prostate cancer [J]. J Natl Cancer Inst, 2000, 92: 1918-1925), under study, analyzed the expression levels of DNA, RNA and protein of tumor samples in different clinical stages, and the result displayed that over-expressed HER2 existed in 25% of patients (UNT tumor) whose prostate cancers were removed through surgery, 59% of patients (TAA tumor) who accepted antiandrogen therapy before surgery and 78% of patients (androgen-independency AI) who failed androgen therapy and generates osseous metastasis.
(5) Lung cancer: the over-expression of HER2 in the lung cancer is mainly related to genetic transcription and posttranscriptional modification. Domestic researches indicated that the over-expression of HER2 mainly happened in the non-small cell lung cancer, mainly in glandular cancer rather than squamous cancer. However, the detection result from 88 hungarian patients suffering the non-small cell lung cancer indicated that the over-expression of HER2 only existed in five cases all of which suffer squamous-cell carcinoma, resulting in different research results. Furthermore, there are different conclusions in the relationship between the over-expression of HER2 in the lung cancer and the cell differentiation degree.
An antibody drug trastuzumab, specific to HER2 targets, has the trade name HERCEPTIN® and is a humanized monoclonal antibody taking HER2 as a target. HERCEPTIN® (trastuzumab) is obtained by mosaicism of a stable domain of non-specific human IgG and an antigenic determinant of mouse anti-HER2 protein IgG through a genetic engineering method, not only has high affinity to an HER2 receptor, but also solves the problem that a mouse-derived antibody is applied to immunogenicity of the human body and can reduce the generation of human anti-mouse antibodies, thus avoiding from being removed by a reticuloendothelial system. In vivo and in vitro experiment researches indicated that the application of HERCEPTIN® (trastuzumab) to down-regulation of expression was capable of retarding the cell growth and remarkably improving its sensitivity to chemoradiotherapy. In 1998, the US Federal Drug Administration (FDA) approved this drug was used for second-line or third-line therapy of HER2 over-expressed metastatic breast cancer, which was also the first and the only humanized monoclonal antibody drug approved for treating HER2/neu protein expressed positive metastatic breast cancer and early-stage breast cancer.
3. Technological Development of Bispecific Antibody
The bispecific antibody is an antibody in which two antigen binding sites in one antibody molecule can bind to two different epitopes respectively.
The antibody drug refers to a biomacromolecular drug prepared by an antibody engineering technology taking a cell engineering technology and a genetic engineering technology as main bodies and has the advantages of high specificity, uniform property, capability of realizing directional preparation against specific targets, etc. The monoclonal antibody is mainly applied to the following three aspects in clinical practice: oncotherapy, therapy of immune diseases and anti-infective therapy. Wherein, the oncotherapy is the most extensive field for monoclonal antibody application at present, and products for oncotherapy in monoclonal antibody products that have entered clinical trial and listed in the market account for about 50%. The oncotherapy by monoclonal antibodies is an immunotherapy for killing target cells by stimulating the immune system against specific targets of pathological cells, in order to enhance the effector function of the antibody, and especially the effect of killing tumor cells; and as concerned in multiple methods that have been tried by people to transform antibody molecules, the bispecific antibody has been one of the development trends for improving the antibody therapy effect and has become the hotspot in the field of antibody engineering researches.
The bispecific antibody for immunotherapy is an artificial antibody containing two kinds of specific antigen binding sites, is capable of building a bridge between the target cell and the functional molecule (cell) and stimulating oriented immunoreaction and has a wide application prospect in immunotherapy of tumors.
4. Preparation of Bispecific Antibody
The bispecific antibody can be obtained by multiple paths, and its preparation methods mainly include a chemical coupling method, a hybrid-hybridoma technique and a genetically engineered antibody preparation method. As concerned in the chemical coupling method, two different monoclonal antibodies are connected together in a chemical coupling manner to prepare a bispecific monoclonal antibody, which is the earliest bispecific monoclonal antibody concept. As concerned in the hybrid-hybridoma technique, the bispecific monoclonal antibody is produced by a cell hybridization method or a ternary hybridoma manner, and these cell hybridomas or ternary hybridomas are obtained through fusion of built hybridomas, or the fusion of the built hybridomas and mouse-derived lymphocytes and could only produce a mouse-derived bispecific antibody, and are thus limited to a great extent in application. With the rapid development of the molecular biological technology, multiple construction modes of humanized bispecific antibodies in genetic engineering have arisen, which are mainly classified into four categories, namely a bispecific micro-antibody, a double-chain antibody, a single-chain bivalent antibody and a multivalent bispecific antibody. At present, there have been several international genetically engineered bispecific antibody drugs that have been entered the clinical trial stage with a better application prospect.
5. Adoptive Immunotherapy of Tumors
As concerned in the adoptive immunotherapy of tumors, mainly comprising immunotherapy of LAK cells, TIL cells, activated T lymphocyte and CIK cells, autologous or allogeneic immunocompetent cells are delivered into the body of a patient after in vitro amplification to directly kill tumor cells, and regulate and enhance the immune function of the organism. However, the immunotherapy can be only used to remove a small number of scattered tumor cells, has a very limited effect on end-stage solid tumors, and is thus usually used as an adjuvant therapy to be combined with conventional methods, such as surgery, chemotherapy and radiotherapy. After a large number of tumor cells are cleared up by the conventional methods, residual tumor cells are removed by the immunotherapy, so that the comprehensive therapy effect on tumors can be improved. Wherein, as a new method for comprehensive therapy of tumors, the adoptive immunotherapy has been widely matched with conventional surgery, radiotherapy, chemotherapy and other cell and molecule therapies and holds great promise in therapy of multiple tumors. However, it should be a more ideal method that one end of the bispecific antibody can bind to a surface antigen CD3 of a cultured immune cell and is delivered into the body along with it, and the other end of the bispecific antibody can well bind to the surface antigen of the tumor cell; and therefore, the bispecific antibody can build a bridge between the tumor cell and the immune cell in the body, so that the immune cells are gathered around the tumor cells to further kill the tumor cells. By this method, the metastasis and diffusion of the tumor cells can be effectively solved, and the defects, such as ‘halfway, easy metastasis and large side effect’ in the three traditional therapy modes, namely surgery, radiotherapy and chemotherapy are overcome.